Cytokines comprise a variety of secreted proteins that regulate cell growth and differentiation. These factors dramatically influence immune and inflammatory responses. Thus understanding the molecular basis of this regulation is likely to provide important insights on the pathogenesis of immune-mediated disease as well as offer new therapeutic targets. We cloned a kinase, Jak3 that is responsible for signaling by a class of cytokines that bind the common gamma chain, gc (IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21). These cytokines regulate lymphocyte development, differentiation, and homeostasis. Mutation of gc or Jak3 results in severe combined immunodeficiency (SCID). A major objective of the laboratory continues to be the understanding the structure and function of Jak3, in vitro and in vivo. One aspect of this project is the identification of new patients with Jak3 mutations. Such patients can give insights into the spectrum of clinical presentations associated with this disorder and also give clues to structure/function. We recently identified one family in which different members exhibited a range of presentations from profound immunodeficiency and severe autoimmunity to being relatively unaffected. We further showed that these Jak3 mutations were associated with oligoclonal expansion of activated T lymphocytes with poor expression of the pro-apoptotic molecule Fas ligand. This suggests that Jak3, likely via IL-2 activation, is essential in humans for lymphoid homeostasis. The data also indicates that the range of clinical presentations associated with Jak3 mutations is much broader than previously appreciated. We also utilize patient mutations to provide new insights into the structure of Jak3. We identified three patient-derived mutations in the amino-terminal FERM (band 4.1, ezrin, radixin and moesin domain). Through the study of these patients, we showed that FERM mutations inhibit Jak/receptor association and inhibit Jak3 catalytic activity. We further showed that the likely explanation for these results was that the FERM and kinase domains associate and that this association positively regulates catalytic activity. Conversely, we showed that a kinase inhibitor could disrupt Jak/receptor association, further substantiating our model of Jak structure. Based on these results, we have initiated a collaboration to obtain more detailed structural information regarding Jak3. Additionally, we had previously identified the docking protein, Gab2, as a Jak3 substrate. In extension of these studies, we have now shown that Gab2 is also an important adapter molecule involved in Fc receptor (FcR)-mediated signaling. We showed that is a unique substrate in that the adapter molecule LAT and the kinases, Syk and Lyn are not required for its phosphorylation. In other collaborative studies, we also found that Jak3 associates with the T-cell receptor and is activated by TCR crosslinking. Of note, the JH4 domain of Jak3 appears to mediate this association. In another collaborative study, an alternatively glycosylated form of the common gamma chain, gc, was identified in myeloid cells. The second major area of investigation is the control of helper T (Th) cell differentiation and the regulation of cell-mediated immunity. Several factors regulate this process including the cytokine IL-12, which activates the transcription factor Stat4. The IL-12R comprises two subunits IL-12R beta 1 and IL-12R beta 2. In a collaborative study, we found that IL-12R beta 2-deficient mice produced no IL-12-induced biological functions, as measured by NK lytic activity, IFN-gamma secretion and Th1 differentiation. In a second study, we have identified the tyrosyl residues critical for recruiting Stat4 to the receptor through mutational of the receptor. These sites are essential for IL-12?s induction of IFN-g and Th1 differentiation. We have also investigated other aspects of IL-12 signaling, including the ability of the IL-12R to couple to p38 MAPK activation and have identified Ser721 of Stat4 as a substrate of this kinase. By mutational analysis we demonstrated that this modification is important in control of transcriptional regulation. By reconstitution of Stat4 deficient lymphocytes with a Ser721Ala mutant of Stat4, we found that phosphorylation of this site is essential for normal IFN-g production and Th1 differentiation. T-bet is a newly described transcription factor that also promotes Th1 differentiation and IFN-g production. We investigated the regulation of T-bet and showed that it is rapidly induced by IFN-g itself. That is, naive T cells express little T-bet whereas TCR occupancy with IFN-g synergistically upregulate T-bet. IFN-g regulation of T-bet is dependent upon Stat1 but is independent of Stat4. Thus, we propose a new model of Th1-differentiation in which IFN-g regulates its own production by induction of T-bet; T-bet/IFN-g cooperate in a feed-forward mechanism of Th1 regulation. It is now appreciated that dendritic cells (DC) and other antigen presenting cells (APCs) regulate Th differentiation. We have found that Stat4 is expressed in activated monocytes and dendritic cells and is highly expressed in monocytes present in rheumatoid synovium. We further demonstrated that the expression of Stat4 in dendritic cells is required for IFN-g production and is involved in autocrine IL-12 responsiveness. Based on these findings, we next investigated the expression of T-bet in DC and macrophages. We demonstrated that IFN-g induces expression of T-bet in these APCs. We propose that the regulation of expression of "Th1"-expressed transcription factors, like T-bet and Stat4, control IFN-g production in APCs and thus serve to regulate Th1 differentiation. We have termed this the "jump start" model of cell-mediated immunity to denote the ability of DC to initiate Th1 differentiation by providing IFN-g. In effort to better understand target genes activated by cytokines, we have utilized microarray technology. Given the therapeutic utility of glucocorticoids, we also investigated the effect of glucocorticoids on activated T-cells. A number of important genes were identified, but we were particularly intrigued, that one of the most highly glucocorticoid-inducible genes is the interleukin-7 receptor (IL-7R). We demonstrated that glucocorticoids can have the unexpected function of enhancing lymphoid survival by upregulating IL-7R. Finally, in collaboration with the Genetics Section of ARB, we have previously shown that the gene underlying an autosomal dominant forms of periodic fever syndromes is TNFRSF1A, the receptor for the cytokine, tumor necrosis factor. We termed this disorder TRAPS, TNFR-associated periodic syndrome. We extended our initial studies to identify six new mutations, including two new, more prevalent, mutations associated with reduced penetrance.